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J Exp Med. Dec 1, 1996; 184(6): 2301–2310.
PMCID: PMC2196367
Articles
Preferential Proliferation of Murine Colony-forming Units in Culture in a Chemically Defined Condition with a Macrophage Colony-stimulating Factor–negative Stromal Cell Clone
Nobuyuki Takakura,* Hiroaki Kodama, Satomi Nishikawa,* and Shin-Ichi Nishikawa*
From the *Department of Molecular Genetics, Faculty of Medicine, Kyoto University, Kyoto 606-01, Japan; and the Institute of Allergy, Research Center Kyoto, Bayer Yakuhin, Ltd., Kyoto 619-02, Japan
Address correspondence to Nobuyuki Takakura, M.D., Department of Molecular Genetics, Faculty of Medicine, Kyoto University, Shogoin-Kawahara-cho 53, Sakyo-ku, Kyoto 606-01, Japan.
Received April 15, 1996; Revised September 16, 1996
Abstract
The establishment of culture conditions that selectively support hematopoietic stem cells is an important goal of hematology. In this study, we investigated the possibility of using for this purpose a defined medium, mSFO2, which was developed for stromal cell–dependent bone marrow cultures. We found that a combination of epidermal growth factor (EGF), the OP9 stromal cell line, which lacks macrophage colony-stimulating factor, recombinant stem cell factor, and the chemically defined medium mSFO2 provides a microenvironment where c-Kit+ Thy-1+/lo Mac-1+/lo B220 TER119 commonβ+ IL-2Rγ+ gp130+ cells are selectively propagated from normal, unfractionated bone marrow cells. This cell population produced an in vitro colony at a very high efficiency (50%), whereas it has only limited proliferative ability in the irradiated recipient. Thus, the cells selected in this culture condition might represent colony-forming units in culture (CFU-c) with short-term reconstituting ability. Transferring this cell population into medium containing differentiation signals resulted in the rapid production of mature myelomonocytic and B cell lineages in vitro and in vivo. The fact that a similar culture condition was created by erb-B2–transduced OP9 in the absence of EGF indicated that EGF exerts its effect by acting on OP9 rather than directly on CFU-c. These results suggested that the balance between self-renewal and differentiation of CFU-c can be regulated by extracellular signals.
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Selected References
These references are in PubMed. This may not be the complete list of references from this article.
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Figures and Tables
Figure 2
Figure 2
Growth kinetics and c-Kit expression of BM cells cultured on OP9 stroma cells with mSFO2 containing EGF and SCF. (A) Unfractionated BM cells (105) from C57BL/6 mice were plated on OP9 stroma cells in mSFO2 containing 30 ng/ml EGF (open circle) (more ...)
Figure 1
Figure 1
Phenotypes of BM cells cultured under various conditions. Unfractionated BM cells (105) from C57BL/6 mouse were plated onto the OP9 stroma cells and cultured in mSFO2 containing 10% FCS (A), 10 ng/ml bFGF, and 100 ng/ml SCF (B), or 30 ng/ml EGF and 100 (more ...)
Figure 3
Figure 3
Growth kinetics of BM cells cultured on the OP9/erbB2 stromal layer and surface phenotype of recovered cells. (A) Unfractionated BM cells (105) derived from C57BL/6 were plated on OP9/erbB2 stroma cells in mSFO2 supplemented with 100 ng/ml SCF. On the (more ...)
Figure 4
Figure 4
Immediate differentiation of cultured c-Kit+Mac-1dull cell in medium-containing serum. Unfractionated BM cells were cultured on OP9/erbB2, as described in the legend to Fig. Fig.3.3. 21 d later, the cells were transferred onto a fresh (more ...)
Table 1
Table 1
Frequency of Colony-forming Cells
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